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. 2012 Jun 19;107(2):315–324. doi: 10.1038/bjc.2012.263

Figure 5.

Figure 5

MLL4 knockdown affects nuclear integrity and expression of growth and angiogenic factors in xenografted tumour. The MLL4 antisense-treated or control mice with colon cancer xenograft were either euthanised to excise the tumour xenograft or perfused with 4% paraformaldehyde at 28th day of treatment. (A) Immunolocalisation of MLL4 and nuclear integrity: Formaldehyde-fixed tumours were sectioned, immunostained with MLL4 antibody and DAPI (for nuclear staining) and analysed under fluorescence microscope. Representative images showing the cellular morphology (DIC), nuclear integrity (DAPI) and MLL4 expression in the control and MLL4 antisense-treated tumours are shown. (B) MLL4 regulates expression of growth and angiogenic factors in vivo: RT–PCR analysis of the RNA extracted from MLL4 antisense-treated tumour tissues by using primers specific to MLL4, VEGF, bFGF, TGFβ2, CD31, HIF1α and β-actin (control). The real-time quantification (relative to GAPDH) is shown in right panel. Bars indicate standard error. (C) The tumour tissue was analysed by ChIP assay using MLL4, H3K4-trimethyl and RNAP II antibodies. The ChIP DNA was analysed by PCR with primers specific to the promoter regions of CD31, TGFβ2 and NRG1. qPCR data are the right panel.